As described in FIG. 1 of PCT International Patent Application PCT/CA98/00096 to Zou and Taylor, (PCT International Publication WO 98/35044 published Aug. 13, 1998, the contents of the entirety of which and the corresponding U.S. patent application Ser. No. 09/355,912, filed Oct. 15, 1999, are incorporated by this reference), acetyl-CoA plays a central role in mitochondrial respiration and plastidial fatty acid biosynthesis. The pyruvate dehydrogenase complex (PDC) oxidatively decarboxylates pyruvate to yield acetyl-CoA.
Plants have both mitochondrial and plastidial isoforms of the PDC (see also U.S. Pat. No. 6,265,636, to Randall et al (Jul. 24, 2001); which is also incorporated in its entirety by this reference). The mitochondrial pyruvate dehydrogenase complex plays a key role in the regulation of acetyl-CoA generation and availability of acetyl moieties for various catabolic and anabolic reactions in plant cells. The mitochondrial PDC is negatively regulated by phosphorylation of the E1α subunit by pyruvate dehydrogenase kinase (PDHK), and positively regulated by dephosphorylation of the PDC by pyruvate dehydrogenase phosphatase (PDCP). Mitochondrially-generated acetyl moieties can find their way into the respiratory tricarboxylic acid (TCA; Krebs) cycle, but also into the plastid compartment where ultimately, acetate units are used by the enzymes of the fatty acid synthesis (FAS) pathway to synthesize fatty acids. These are eventually incorporated into membrane and also storage glycerolipids.
Zou and Taylor also disclose the identification, isolation and characterization of the pyruvate dehydrogenase kinase (PDHK) (gene and cDNA) sequence from the model plant system Arabidopsis thaliana and the utilization of this sequence in the genetic manipulation of plants. Also disclosed is a vector containing the full-length PDHK sequence or a significant portion of the PDHK sequence from Arabidopsis, in an anti-sense orientation under control of either a constitutive or a seed-specific promoter, for re-introducing into Arabidopsis or for introducing into other plants. Zou and Taylor also provided a method to construct a vector containing the full-length PDHK sequence or a significant portion of the PDHK sequence from Arabidopsis, in a sense orientation under control of either a constitutive or a seed-specific promoter, for re-introducing into Arabidopsis or for introducing into other plants. Also disclosed were methods for modifying Arabidopsis and other plants to change their seed oil content, average seed weight or size, respiration rate during development, vegetative growth characteristics, flowering time or patterns of generative growth, and the period required to reach seed maturity.
As disclosed in, for example, Zou and Taylor, respiration, which involves the consumption of O2 and the catabolism of sugar or other substrates to produce CO2, plays a central role in the process of plant growth in providing reducing equivalents, a source of energy and an array of intermediates (carbon skeletons) as the building blocks for many essential biosynthetic processes. The intermediate products of respiration are necessary for growth in meristematic tissues, maintenance of existing phytomass, uptake of nutrients, and intra- and inter-cellular transport of organic and inorganic materials. Respiration is important to both anabolic and catabolic phases of metabolism.
The pyruvate dehydrogenase complex (PDC) is a particularly important site for regulation of plant respiration. Modification of PDC activity through manipulation of PDHK expression can result in a change in the production or availability of mitochondrially-generated acetyl-CoA or a change in the respiration rate. These changes may in turn affect seed oil content, average seed weight or size, respiration rate during development, vegetative growth characteristics, flowering time or patterns of generative growth, and the period required to reach seed maturity.
Many examples exist of successful modifications to plant metabolism that have been achieved by genetic engineering to transfer new genes or to alter the expression of existing genes, in plants. It is now routinely possible to introduce genes into many plant species of agronomic significance to improve crop performance (e.g., seed oil or tuber starch content/composition; meal improvement; herbicide, disease or insect resistance; heavy metal tolerance; etc.) (Somerville, 1993; Kishore and Somerville, 1993; MacKenzie and Jain, 1997).
The Brassica genus includes Arabidopsis thaliana. The Brassicaceae family is comprised of a large and diverse group of plant species which are economically very important throughout the world. Three diploid Brassica species (B. rapa, B. oleracea and B. nigra) have hybridized in different combinations to give rise to the three amphidiploid species (B. napus, B. juncea, and B. carinata). Other Brassica species include B. oleifera, B. balearica, B. cretica, B. elongate, B. tourneforii, and B. biennis. B. napus and B. rapa have been improved through breeding programs and are now cultivated as canola crops.
It would be an improvement in the art to isolate and sequence the PDHK gene from various useful species of plants of the Brassicaceae.